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Pigs immunized with a novel E2 subunit vaccine are protected from subgenotype heterologous classical swine fever virus challenge

View Article: PubMed Central - PubMed

ABSTRACT

Background: Classical swine fever (CSF) or hog cholera is a highly contagious swine viral disease. CSF endemic countries have to use routine vaccination with modified live virus (MLV) vaccines to prevent and control CSF. However, it is impossible to serologically differentiate MLV vaccinated pigs from those infected with CSF virus (CSFV). The aim of this study is to develop a one-dose E2-subunit vaccine that can provide protection against CSFV challenge. We hypothesize that a vaccine consisting of a suitable adjuvant and recombinant E2 with natural conformation may induce a similar level of protection as the MLV vaccine.

Results: Our experimental vaccine KNB-E2 was formulated with the recombinant E2 protein (Genotype 1.1) expressed by insect cells and an oil-in-water emulsion based adjuvant. 10 pigs (3 weeks old, 5 pigs/group) were immunized intramuscularly with one dose or two doses (3 weeks apart) KNB-E2, and 10 more control pigs were administered normal saline solution only. Two weeks after the second vaccination, all KNB-E2 vaccinated pigs and 5 control pigs were challenged with 5 × 105 TCID50 CSFV Honduras/1997 (Genotype 1.3, 1 ml intramuscular, 1 ml intranasal). It was found that while control pigs infected with CSFV stopped growing and developed high fever (>40 °C), high level CSFV load in blood and nasal fluid, and severe leukopenia 3–14 days post challenge, all KNB-E2 vaccinated pigs continued to grow as control pigs without CSFV exposure, did not show any fever, had low or undetectable level of CSFV in blood and nasal fluid. At the time of CSFV challenge, only pigs immunized with KNB-E2 developed high levels of E2-specific antibodies and anti-CSFV neutralizing antibodies.

Conclusions: Our studies provide direct evidence that pigs immunized with one dose KNB-E2 can be protected clinically from CSFV challenge. This protection is likely mediated by high levels of E2-specific and anti-CSFV neutralizing antibodies.

No MeSH data available.


Related in: MedlinePlus

Production and characterization of recombinant CSFV E2 protein. a SDS-PAGE analysis. Lane 1, E2 protein (1 μg) treated with Laemmli sample buffer with addition of reducing reagent β-mercaptoethanol (β-ME); Lane 2, E2 protein (1 μg) treated with Laemmli sample buffer without β-ME. b Western blot of purified E2 protein. Lane 1, E2 protein (1 μg) treated with β-ME; Lane 2, E2 protein (50 ng) purified and stored under non-reducing conditions. E2-specific Mab WH211 was used for the western blot
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Fig1: Production and characterization of recombinant CSFV E2 protein. a SDS-PAGE analysis. Lane 1, E2 protein (1 μg) treated with Laemmli sample buffer with addition of reducing reagent β-mercaptoethanol (β-ME); Lane 2, E2 protein (1 μg) treated with Laemmli sample buffer without β-ME. b Western blot of purified E2 protein. Lane 1, E2 protein (1 μg) treated with β-ME; Lane 2, E2 protein (50 ng) purified and stored under non-reducing conditions. E2-specific Mab WH211 was used for the western blot

Mentions: CSF virus E2 gene from the HCLV was cloned into the recombinant baculovirus and recombinant E2 protein was produced using High Five insect cells. The culture medium was then collected by centrifugation and subjected to protein purification with Ni-NTA agarose beads. As shown in Fig. 1a, pure recombinant E2 protein was obtained from the condition medium. Under reducing conditions, recombinant E2 protein appeared to be 45 kDa, while the native E2 protein mainly existed as homodimer under non-reducing conditions with a molecular weight of ~90 kDa (Fig. 1a). The dimerization of recombinant E2 protein expressed in insect cells was further confirmed by western blot analysis with anti-E2 mAb WH211. It is worth noting that although the amount of E2 dimer protein loaded in lane 2 on Fig. 1b was only 1/20 of the monomeric E2 (50 ng vs. 1 μg), E2 dimers seem to have a higher affinity to WH211 than did the E2 monomer as evidenced by the difference in intensity of the E2 bands on western blot (Fig. 1b).Fig. 1


Pigs immunized with a novel E2 subunit vaccine are protected from subgenotype heterologous classical swine fever virus challenge
Production and characterization of recombinant CSFV E2 protein. a SDS-PAGE analysis. Lane 1, E2 protein (1 μg) treated with Laemmli sample buffer with addition of reducing reagent β-mercaptoethanol (β-ME); Lane 2, E2 protein (1 μg) treated with Laemmli sample buffer without β-ME. b Western blot of purified E2 protein. Lane 1, E2 protein (1 μg) treated with β-ME; Lane 2, E2 protein (50 ng) purified and stored under non-reducing conditions. E2-specific Mab WH211 was used for the western blot
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5016919&req=5

Fig1: Production and characterization of recombinant CSFV E2 protein. a SDS-PAGE analysis. Lane 1, E2 protein (1 μg) treated with Laemmli sample buffer with addition of reducing reagent β-mercaptoethanol (β-ME); Lane 2, E2 protein (1 μg) treated with Laemmli sample buffer without β-ME. b Western blot of purified E2 protein. Lane 1, E2 protein (1 μg) treated with β-ME; Lane 2, E2 protein (50 ng) purified and stored under non-reducing conditions. E2-specific Mab WH211 was used for the western blot
Mentions: CSF virus E2 gene from the HCLV was cloned into the recombinant baculovirus and recombinant E2 protein was produced using High Five insect cells. The culture medium was then collected by centrifugation and subjected to protein purification with Ni-NTA agarose beads. As shown in Fig. 1a, pure recombinant E2 protein was obtained from the condition medium. Under reducing conditions, recombinant E2 protein appeared to be 45 kDa, while the native E2 protein mainly existed as homodimer under non-reducing conditions with a molecular weight of ~90 kDa (Fig. 1a). The dimerization of recombinant E2 protein expressed in insect cells was further confirmed by western blot analysis with anti-E2 mAb WH211. It is worth noting that although the amount of E2 dimer protein loaded in lane 2 on Fig. 1b was only 1/20 of the monomeric E2 (50 ng vs. 1 μg), E2 dimers seem to have a higher affinity to WH211 than did the E2 monomer as evidenced by the difference in intensity of the E2 bands on western blot (Fig. 1b).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Classical swine fever (CSF) or hog cholera is a highly contagious swine viral disease. CSF endemic countries have to use routine vaccination with modified live virus (MLV) vaccines to prevent and control CSF. However, it is impossible to serologically differentiate MLV vaccinated pigs from those infected with CSF virus (CSFV). The aim of this study is to develop a one-dose E2-subunit vaccine that can provide protection against CSFV challenge. We hypothesize that a vaccine consisting of a suitable adjuvant and recombinant E2 with natural conformation may induce a similar level of protection as the MLV vaccine.

Results: Our experimental vaccine KNB-E2 was formulated with the recombinant E2 protein (Genotype 1.1) expressed by insect cells and an oil-in-water emulsion based adjuvant. 10 pigs (3 weeks old, 5 pigs/group) were immunized intramuscularly with one dose or two doses (3 weeks apart) KNB-E2, and 10 more control pigs were administered normal saline solution only. Two weeks after the second vaccination, all KNB-E2 vaccinated pigs and 5 control pigs were challenged with 5 × 105 TCID50 CSFV Honduras/1997 (Genotype 1.3, 1 ml intramuscular, 1 ml intranasal). It was found that while control pigs infected with CSFV stopped growing and developed high fever (>40 °C), high level CSFV load in blood and nasal fluid, and severe leukopenia 3–14 days post challenge, all KNB-E2 vaccinated pigs continued to grow as control pigs without CSFV exposure, did not show any fever, had low or undetectable level of CSFV in blood and nasal fluid. At the time of CSFV challenge, only pigs immunized with KNB-E2 developed high levels of E2-specific antibodies and anti-CSFV neutralizing antibodies.

Conclusions: Our studies provide direct evidence that pigs immunized with one dose KNB-E2 can be protected clinically from CSFV challenge. This protection is likely mediated by high levels of E2-specific and anti-CSFV neutralizing antibodies.

No MeSH data available.


Related in: MedlinePlus